The Effect of essential oil of Oregano (Origanum vulgare) on the in vitro growth of the bacteria Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus

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The Effect of essential oil of Oregano (Origanum vulgare) on the in vitro growth of the bacteria Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus
Eric T. Rueda

Department of Biological Sciences

Saddleback College

Mission Viejo, CA 92692

Oregano oil is a very potent source of the antimicrobial substance Carvacrol. Three bacteria, Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, were exposed to this natural active chemical. They were incubated at 37.0°C and data was collected by subtracting the total area of inhibition (including the area occupied by the chad) and subtracting the area of the oregano (Origanum vulgare) oil dipped chad. Results were calculated as the average area of inhibition and graphed, however, they did not stand up to par with previous experiments. The growth of the bacteria was mildly effected by the presence of the oregano oil.
The immune system is a complex defense system constantly fighting off infection and assisting the body as it strives to achieve homeostasis. These functions will continue regardless of the macroscopic environment that we consciously expose ourselves to. The body thrives under control of the brain and its collective use of complex systems, such as the nervous, lymphatic, and digestive systems. Electrical and chemical messages relay from exterior or interior stimuli and the body will react accordingly.

In the case of an unwanted microbe or bacterium within our body, the immune system works to destroy this parasite by surface markers, cytokines, phagocytosis, or antibodies (Campbell et al., 2008). Multiple cells and tissues are affected by these changes; (this sentence needs to be re-worded) such as if the body has no memory of the parasite, then an acquired immunity develops. This allows for cells to retain information of the parasite so that it can be destroyed faster and more efficiently. Our body is good at (use better word choice) managing a broad spectrum of infection, however, when a parasite (Why is the word parasite being used?? How about pathogen or microorganism?) reproduces too rapidly within our body or it is extremely unfamiliar, we may be working to catch up to it to destroy it. If this is the case, then one may seek medical attention.

Medical practice has developed and grown exponentially over an incredible amount of time. Dating back to before 1400 B.C. (McCall et al., 2003), society made use of simple, yet misunderstood blood letting techniques to remove excess bile. Currently, the procedure of a standard infection follows that a prescription medication is administered to the patient in hope, and through multiple trials and experience, that the parasite will be vanquished. Prescription medication is used regularly and has become a multibillion dollar industry (Wechsler, 2010). Through private investors and government funding, this field targets FDA approval of microbiological based medicines. Scientists draw inspiration from nature to fuel cognition and fabricate new medications and medical techniques. Over time, plants and fungi have developed adaptations to survive extreme environments and to keep them free of certain microbes. In particular, the oregano plant (Origanum vulgare) (Morris 2005) makes use of two phenol compounds: Carvacrol (C10H14O) and Thymol (C10H14O) (Preuss et al. 2005). The main body constructed in a benzene ring with different appendage hydroxyl groups. Carvacrol, specifically, has been shown through many in vitro experiments to be a strong antibacterial compound (Nutraceuticals, 2001; Preuss et al., 2005; Ott et al., 2008; Sarac et al., 2008; Erickson, 2009; Wong et al., 2008; Patrone, 2010). Carvacrol has repeatedly displayed antimicrobial activity against related bacteria found in animals and plants, such as strains of Aspergillus, Streptococcus, Bacillus, and Candida. These concentrations are as low as 0.625 mg/mL (Can Baser, 2008). Carvacrol inhibits bacteria by creating an ionic imbalance on the cell membrane by allowing K+ ions to leak from the cell resulting in unstable cytoplasmic pH levels and osmotic pressure. This membrane now leaks ATP due to the breakdown of the cellular membrane leaving the cell unable to function. Carvacrol will also inhibit ATPase, the enzymatic active-transport pump located on the cellular membrane which functions to regulate proton distribution between the cytoplasm and extracellular fluid. (Campbell et al., 2008) With such a long standing and well documented history of antimicrobial qualities, the objective of the study was to examine if oregano oil would inhibit the growth of three bacteria: Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus. It is hypothesized that the growth of the bacteria would be greatly effected the presence of the oil.
Materials and Methods
Forty nutrient agar plates were prepared using Difco nutrient agar. Three bacteria: Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus, were cultured and provided by the Saddleback Biology Department. Sterile water was used as the control.

Supplies and methods were completed using aseptic technique; gloves and eye shield were dawned and all materials used were from sterile packaging or had been autoclaved. Serological pipettes were used to transfer 0.3 mL of E. coli to the nutrient agar plates. A glass rod spreader was used to distribute the bacteria evenly around the petri dish. After each use, the glass spreader was dipped in 95% ethanol, passed through a Bunsen burner flame before being used on the next agar plate. The procedure was repeated for S. typhimurium, S. aureus, and water control plates.

Three chad disks were dipped in the oregano oil and placed onto each agar plate using forceps. The brand Nature’s Answer alcohol-free oregano oil, purchased from The Vitamin Shoppe in Mission Viejo, California, was combined with olive oil and contained active ingredient Carvacrol at 7.0 mg per 0.2 mL of oil. This dropper bottle was purchased from The Vitamin Shoppe in Mission Viejo, California. Forceps were dipped in 95% ethanol and passed through a Bunsen burner flame before handling each disk. All agar plates were incubated at 37.0°C for 48 hours at Saddleback College.

Data was collected by measuring the entire diameter of the chad and area of inhibition. Measurements were taken using a metric ruler (millimeters) and values were recorded to the tenths. Area of inhibition of one chad disk was calculated by subtracting the chad disk diameter, 7.0 mm, from the inhibition diameter. The summation of these values was then divided by the total number (n=30) to obtain the mean diameter of inhibition. Statistical test ANOVA was run comparing the bacterial growth to each other.

The oregano oil had a similar effect on the mean area of inhibition on each bacteria. An ANOVA statistical test was used to analyze the significance of the area of inhibition of all bacteria in relationship to each other. A significant P-Value of 1.04E-09 was found between the groups. The Bonferroni Correction Test with MS Residual of 14.63211111 and DS Residual of 3.0, and a confidence interval of 95% was run to compare each bacteria to each other. Compared to one another the bacteria showed no statistical significance, with a P-Value > 0.05 (Figure 1).
Delete title on figure and the horizontal lines, X-axis label?

Figure 1. The comparison of inhibition of growth of bacteria (n=30) Staphylococcus aureus, Escherichia Coli, and Salmonella typhimurium against oregano (Origanum vulgare) oil (Carvacrol at 7.0 mg per 0.2 mL). Control was water and showed zero inhibition. P-Value was statistically insignificant (> 0.05) (Bonferroni Correction Test) and bacteria are graphed with a 95% confidence interval. S.aureus standard error of 0.158832, E.coli standard error of 0.172239, and S.typhimurium standard error of 0.229767.

The expected results are not supported by the outcome of the collected data. Through previous studies such as Can Baser (2008) and Wong (2008), oregano oil inhibited the growth of these three bacteria significantly. An important factor, in this study, that may have caused a deviation from the expected results is the low concentration of Carvacrol in the sample tested. A small range of inhibition was due to the presence of Carvacrol, however Carvacrol did not have a statistically significant effect on the growth of the three bacteria Staphylococcus aureus, Escherichia Coli, and Salmonella typhimurium.

This s a very short discussion – is this worth 24 points?

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Review Form

Department of Biological Sciences

Saddleback College, Mission Viejo, CA 92692

Author (s): Eric T. Rueda

Title: The Effect of essential oil of Oregano (Origanum vulgare) on the in vitro growth of the bacteria Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus

Researchers investigated the antibiotic properties of oil produced by the Oregano plant, Origanum vulgare. Carvacrol is one of the antibiotic compounds found in the essential oil of Oregano. The researchers found that the Carvacrol did not have a significant inhibitory effect on the 3 types of bacteria used.

General Comments

I thought that the paper had some interesting points. A better choice of scientific dialogue needs be used in a few areas that I highlighted. I provided some suggestions and rewrote some areas.

Technical Criticism

Overall, organization was good. Some minor mechanical and grammatical errors have been corrected. The graph needs to have the horizontal lines removed from the background.

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